Aeromonas salmonicida is a non-motile, facultatively anaerobic, Gram-negative rod-shaped bacterium of the Vibrionaceae family. It is the etiological agent of salmonid furunculosis, a disease capable of causing serious losses in both cultured and wild stocks of salmonids such as the Pacific salmon (Oncarhynchus sp.), rainbow trout (O. mykiss), and the Atlantic salmon (Salmo salar).
The use of antibiotics to treat furunculosis disease in fish has encountered some significant problems, primarily the development of antibiotic resistance in the causative microorganisms. Extensive research has therefore been performed in recent years to develop vaccines to prevent this disease. Vaccines previously developed for the prevention of furunculosis have included killed bacteria (bacterins) administered either orally, by injection, or by immersion or spraying (Klontz and Anderson, 1970; Paterson and Fryer, 1974; Michel, 1979; McCarthy et al., 1983; Johnson and Amend, 1984; Olivier et al., 1985; Tatner, 1987). So far, injection is the only route of administration which has provided reasonable immunity against this disease in fish. Injectable vaccines are not as practical or desirable as immersion vaccines for vaccinating large salmonid populations because injections are time-consuming, expensive and cause physiological stress in the fish. In spite of a strong commercial demand and extensive research, an effective vaccine for furunculosis that does not require administration by injection, has eluded researchers since the first oral vaccine trials of Duff (Duff, 1942).
Recently, the production of live, attenuated bacterial vaccines has received a great deal of attention. Live vaccines generally produce a more effective immunity compared to vaccines based on killed pathogens or bacterial components. Furthermore, live vaccines may require a smaller dose than killed pathogens or single bacterial component vaccines and may be less expensive to produce. The production of bacterial component vaccines requires potentially complex purification steps while the production of killed pathogen vaccines requires very careful control and assessment of the product to ensure that no live pathogens remain.
Some live, attenuated vaccines have been developed, primarily for the prevention of enteric salmonellosis (Hoiseth and Stocker, 1981; Sigwart et al., 1989). These vaccines have employed a variety of compromising mutations, most of which would be difficult to reproduce in A. salmonicida which is nutritionally fastidious and physiologically unexplored. In some instances, spontaneous antibiotic resistant mutants of bacterial pathogens have been demonstrated to be both avirulent and immunogenic (Brubaker and Surgella, 1962; Cipriano and Starliper, 1982; Bolin et al., 1985; Norqvist et al., 1989) and may offer some potential as vaccines.
However, a major concern with the use of these spontaneous attenuated mutants as vaccines is the frequency of reversion of such mutants to the virulent form. A single point mutation may revert with a frequency as high as 10.sup.-7, resulting in the conversion of the attenuated strain back to the virulent pathogen. Because of this high reversion frequency, such mutants could not safely be released into the environment as a live vaccine and would therefore be unsuitable for use in large scale fish farming.
Recently, genetically engineered attenuated mutants of A. salmonicida (Vaughan et al., 1990) and another fish pathogen, Vibrio anguillarum, have been produced (Singer et al., 1991; Norqvist et al., 1989). Genetically engineered attenuated strains have a number of potential drawbacks as vaccines, such as the inclusion of antibiotic resistance markers in the engineered organisms which could be spread to other pathogens. The potential for reversion to virulent forms also needs--to be considered, especially where the avirulence is caused by (readily reversible) transposon mutagenesis. Thus, extensive research will be required to ensure the safety and predictability of a genetically engineered attenuated form of a pathogenic organism before it can be released into the environment.
In attempts to elucidate aspects of A. salmonicida which might provide insight into the development of effective vaccines, a number of studies have addressed the virulence properties of the organism. Among other aspects of the organism's physiology and metabolism that may influence virulence, the A-layer has received considerable attention (Trust et al., 1982; Trust et al., 1983; Ellis et al., 1988; Kay et al., 1986; McCarthy et al., 1983; Olivier et al., 1985). Virulent A. salmonicida strains possess a distinct surface layer (the A-layer) external to the outer membrane. This A-layer is organized as a tetragonal array and is primarily composed of a 50,000 M.sub.r protein termed the A-protein, associated with lipopolysaccharide (LPS). The layer has been shown to completely envelop A. salmonicida cells, and to physically protect underlying outer membrane components from bacteriophage, complement and proteolytic digestion. LPS has been shown to be essential in maintenance of A-layer integrity since LPS deficient (LPS.sup.-) mutants are invariably A-layer deficient (A.sup.-) as well.
Studies with a variety of isogenic strains (A.sup.+ LPS.sup.+, ALPS.sup.+, A.sup.- LPS.sup.-) indicate that the A-protein is largely responsible for the hydrophobic nature of the cell surface of virulent strains. A-layer deficient strains are rapidly cleared from fish and, when present, the A-layer enhances adsorption of bacteria to macrophages and appears to confer resistance to the cellulolytic activity of these phagocytes.
It has been suggested that the A-layer is a primary virulence factor in A. salmonicida, aiding in survival of the pathogen and enhancing its spread (Trust et al., 1982; Trust et al., 1983; Kay et al., 1986). Trust et al. (1982) have stated that in order to resist infection by A. salmonicida, "fish immunelogical defense mechanisms likely need to recognize and effectively respond to this A-protein." The A-protein has therefore been considered an obvious candidate for inclusion in vaccines.
Olivier et al. (1985) compared the immunogenicity of virulent and avirulent A. salmonicida strains by injection into fish and rabbits. The results of this study showed that avirulent strains were inferior immunegens and it was suggested that the superior immunogenic properties of the virulent strains were conferred by the A-layer which was believed to be absent from the avirulent strains. McCarthy et al. (1983) demonstrated passive immunity to A. salmonicida in fish. This was achieved by raising antisera in rabbits against strains of A. salmonicida, including an A.sup.- variant. Fish were injected with these antisera and the protection afforded by each antiserum was assessed by challenge with virulent A. salmonicida. Only antisera raised against A.sup.+ strains of the organism were found to confer passive immunity, suggesting that this immunity was due to anti-protein-A antibodies. Furthermore, these researchers also tested a number of bacterin vaccines, including an A.sup.- variant, by immersion of fish in the bacterin vaccine followed by challenge with virulent A. salmonicida. Only bacterins made from A.sup.+ A-salmonicida were effective in protecting the fish, leading the authors to conclude that "[t]he A-protein of A. salmonicida is . . . needed to confer protection with bacterins" (McCarthy et al.,1983). Thus the immunogenicity of spontaneous, A-layer deficient, avirulent mutants has generally been found to be insufficient for their use as effective vaccines.
Cipriano and Starliper (1982) described a spontaneous, avirulent mutant that did confer protection against infection when administered by immersion, however the nature of this mutation is unknown and this finding has not been reproduced by others. It is also important to reiterate the dangers of reversion to virulence inherent in such spontaneous mutants.
Despite various attempts to formulate reliable vaccines against A. salmonicida infection which can be administered to fish orally or by immersion, suitable vaccines have not been developed to date.
It is an object of this invention to provide an improved live, attenuated vaccine that can reduce the susceptibility of fish to infection by A. salmonicida.
It is a further object of this invention to provide an improved live attenuated vaccine for A. salmonicida that can be released into the environment without significant potential for reversion of the organism into the virulent progenitor form.
Finally, it is an object of this invention to provide a live attenuated vaccine that may be administered to fish orally or by immersion, yet has efficacy comparable or superior to a vaccine administered by peritoneal injection.